The goal of the proposed work is to develop a novel method for delivering biologically interesting nucleoside triphosphates into cells that would bypass the (deoxy)nucleoside, (d)NMP and NDP kinases normally needed to convert nucleosides into nucleoside triphosphates. Nucleosides are an extraordinarily important class of antiviral and anticancer therapeutics. The biologically active form of nucleosides is generally the nucleoside triphosphate, and their activity results from inhibition of DNA or RNA polymerases and/or incorporation into DNA or RNA. Unfortunately, the kinases involved in converting a nucleoside to a nucleoside triphosphate generally exhibit substantial specificity towards both the base and sugar. Consequently, a large number of potentially therapeutic nucleosides are completely useless as polymerase inhibitors because they are not converted to the biochemically active triphosphate. To overcome this problem, a novel prodrug approach will be developed that will allow for the delivery of masked nucleoside triphosphate mimics into cells. Upon entering the cells, unmasking will be initiated by cellular enzymes to liberate the nucleoside triphosphate mimic. In these initial studies, HIV reverse transcriptase will be targeted since a number of anti-HIV drugs are not efficiently phosphorylated by cellular kinases and reverse transcriptase readily tolerates modification of the triphosphate. The methods developed, however, are designed to be broadly applicable to virtually any nucleoside triphosphate and most DNA (or RNA) polymerases. To accomplish this goal, two specific aims will be addressed: (1) Develop highly efficient and robust syntheses of nucleoside triphosphate prodrugs and characterize their chemical properties. (2) Optimize the biochemical properties of the prodrugs so that they efficiently enter cells and are unmasked to generate the triphosphate. The results of these studies will be used to direct the synthesis of prodrugs of 3'- azido-3'-deoxythymidine triphosphate that will be tested as inhibitors of HIV. PUBLIC HEALTH RELEVANCE: Nucleoside analogues have proven to be an extremely powerful class of both anti-cancer and anti- viral chemotherapeutics. However, the specificity of the kinases required for converting a nucleoside into the medically relevant nucleoside triphosphate often limits the utility of potentially interesting nucleosides. Having a generally applicable method for directly delivering nucleoside triphosphate mimics into cells could greatly expand the utility of nucleoside analogues and potentially lead to new treatments of both viral infections and cancer.